(633f) In Situ Crosslinked Endosomolytic Polymer Vesicles for Delivery to Cytosolic Antigen Processing and Immune Surveillance Machinery

Subunit vaccines represent a safer, more scalable, and molecularly defined alternative to most current vaccines that are based on attenuated or inactivated pathogens. However, subunit antigens are weakly immunogenic, requiring the addition of immunostimulatory vaccine adjuvants that enhance and shape antigen-specific immune responses. The ongoing discovery of pattern recognition receptors (PRRs) is informing the rational design of molecularly defined adjuvants, including cyclic dinucleotides (CDNs), which trigger activation of the cytosolic stimulator of interferon genes (STING) pathway. However, inefficient cytosolic delivery remains a significant barrier to the development of this emerging class of vaccine adjuvant. To address this challenge, we have developed polymeric vesicles that actively enhance endosomal escape of encapsulated protein antigens and CDNs to cytosolic antigen processing and immune surveillance pathways.

Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize poly[(ethylene glycol)-b-(butyl methacrylate-co-diethylamino ethyl methacrylate-co-pyridyl disulfide ethyl methacrylate)] (PEG-b-DBP) amphiphilic diblock polymers that self-assemble into pH-responsive vesicles with d < 100 nm in aqueous conditions with neutral surface charge. Notably, pyridyl disulfide (PDS) moieties were exploited for post-assembly in situ crosslinking of the vesicle membrane via partial reduction of disulfide bonds with dithiothreitol (DTT). Both crosslinked and non-crosslinked particles exhibited a drop in hydrodynamic radius as pH was reduced from 7.4 to 5.8, corresponding to particle disassembly, release of encapsulated cargo, and transition to a membrane destabilizing state. Crosslinking was found to increase the effective average molecular weight of polymer chains, resulting in enhanced pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with a five-fold increase in hemolysis at pH 5.8 observed at the optimal crosslink density.

Vesicles were shown to encapsulate a diversity of water-soluble cargo, including ovalbumin, a 45kD protein antigen, and the CDN cyclic di-GMP, with encapsulation efficiencies exceeding 20%. Optimized particle formulations were shown to increase cross presentation of ovalbumin `on major histocompatibility complex class (MHC-I) in murine dendritic cells, with crosslinked particles promoting significantly higher class I presentation relative to the uncrosslinked variant. Additionally, vesicles dramatically enhanced delivery of CDNs to the STING pathway in macrophage cell lines, with crosslinked particles inducing a four-fold higher type-I interferon response relative to uncrosslinked analogues. Collectively, these data demonstrate that endosomolytic polymer nanoparticles provide a versatile vaccine delivery platform for targeting cytosolic immunoregulatory machinery.